Name: an immunofluorescence-based method to quantify replication stress in cancer cells
Uploaded: 2024-04-30T12:28:23.000+00:00
Duration: 1 min 13 s
Description: NOTE: The ovarian cancer cell line, OVCAR3, was used in these steps, but this protocol is broadly applicable to multiple other cell lines, including those ...

an immunofluorescence-based method to quantify replication stress in cancer cells

An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells

Add 1 milliliter of cell suspension on poly-L-lysine coverslips placed in the wells of a 24-well plate, and grow the cells in the culture medium under standard conditions. After one population doubling, aspirate the existing media from the plate and pulse the cells with 10 micromolar IdU for the two subsequent population doublings.
To harvest the cells, replace the medium with ice-cold 0.5% PBSTx on ice for 5 minutes. For fixation of cells, aspirate PBSTx, and incubate the cells for 15 minutes with 3% paraformaldehyde at room temperature. After 3 to 4 washes with PBS, keep fixed cells at 4 degrees Celsius until further use.
After fixation, permeabilize the cells using 0.5% PBSTx on ice for 5 minutes, ensuring to cover the entire coverslip. After washing the cells 3 to 4 times with 1 milliliter of 0.2% PBST at room temperature, aspirate the PBST, and block the samples, using 5% BSA made in PBS for 30 minutes at room temperature.
For immunostaining, prepare a humidified chamber by putting a wet paper towel on a flat-bottom Tupperware. Cover the 24-well plate lid with parafilm. Place it in the humidified chamber, and lay down the coverslips on the plate lid. Add 60 microliters of diluted anti-BrdU primary mouse antibody, on the top of the coverslip.
Alternatively, to reduce the antibody from drying and use less volume, place a drop of diluted antibody on the parafilm and flip the coverslip onto it. Then, incubate for 1 hour at 37 degrees Celsius. After incubation, aspirate the primary antibody.
Return the coverslips back to a 24-well plate and wash them with 0.2% PBST 4 times. Add diluted secondary antibody on the coverslip, as demonstrated earlier, and incubate for one hour in the dark at room temperature. Label a microscope slide, and mount the coverslip on the slides with DAPI mounting medium. Store slides in the dark at room temperature for 24 hours if the mounting medium needs to be cured or hardened.

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NOTE: The ovarian cancer cell line, OVCAR3, was used in these steps, but this protocol is broadly applicable to multiple other cell lines, including those derived from non-ovarian sources. A schematic of the protocol is shown in&#160;Figure 1.
1. Plating the cells
<ol>
	<li>Make poly-L-lysine coated coverslips.
	<ol>
		<li>Add autoclaved 12 mm diameter coverslips to a 50 mL conical tube with poly-L-lysine solution, and place on a rocker for 15 min.</li>
		<li>Aspirate the solution in a tissue culture hood. Wash the coverslips by adding sterile water and place the tube containing the coverslips back on the rocker for 5 min. Repeat this wash step three times.</li>
		<li>Spread the coated coverslips on a sterile dish and aspirate any remaining water. Let the coverslips dry in the tissue culture hood for 1 h or until no water droplets remain. Once dry, seal the dish with parafilm, and place at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Place one poly-L-lysine-coated coverslip in each well of a 24-well plate. The use of poly-lysine coverslips is critical to prevent the detachment of the cells from the coverslips during the pre-extraction step.</li>
	<li>Trypsinize OVCAR3 cells once they reach 70%-80% confluency.
	<ol>
		<li>To trypsinize a 10 cm culture dish that is 70%-80% confluent, aspirate the medium from the plate, and wash with 5-7 mL of phosphate-buffered saline (PBS).</li>
		<li>Aspirate the PBS, add 1 mL of 0.25% trypsin, and place the dish in a 37 &#176;C incubator for 8-10 min, or until cells lift off from the bottom of the plate.</li>
		<li>Collect the cells with 5-10 mL of medium and add to a conical tube.</li>
		<li>Count the cells manually with a hemocytometer using standard procedures.</li>
	</ol>
	</li>
	<li>Make a dilution of 25,000 cells/mL and add 1 mL of the cells on poly-L-lysine coverslips placed in the wells of 24-well plates. For any other cell line, determine a number such that the cells are about 70%-80% confluent after three population doublings.</li>
	<li>Grow the cells in the culture medium under standard conditions.</li>
</ol>
2. Pulsing cells with IdU
<ol>
	<li>For the cells to properly spread onto the coverslips, one population doubling is enough. After one population doubling, pulse the cells with 10 &#181;M 5-iodo-2&#39;-deoxyuridine (IdU) (see&#160;Table of Materials&#160;on how to reconstitute IdU) for the two subsequent population doublings.&#160;&#160;&#160;&#160; 
	NOTE: We have tried different thymidine analogs, including bromodeoxyuridine (BrdU), 5-chloro-2&#8242;-deoxyuridine (CIdU), and IdU. Amongst the three analogs, IdU gives the best signal-to-noise ratio. Comparative images of cells pulsed with the three different analogs are shown in&#160;Figure 2. We recommend the use of two negative controls: (i) a no IdU pulsed sample, and (ii) a no primary antibody control. If the formation of ssDNA needs to be assessed due to a given treatment, we recommend adding the drug after the first round of IdU doubling.</li>
	<li>After pulsing with IdU for two population doublings, harvest the cells for imaging.
	<ol>
		<li>Replace the medium with ice-cold 0.5% PBSTx (PBS + 0.5% Triton X-100) on ice for 5 min. This pre-extraction step helps to release cytoplasmic and non-chromatin-bound proteins, leaving chromatin-bound proteins intact.&#160;Certain cell lines can easily peel off during pre-extraction. In such a scenario one can use 0.5% CSK buffer (10 mM PIPES (pH 6.8),100 mM sodium chloride (NaCl), 300 mM sucrose, 3 mM magnesium chloride (MgCl2), 1 mM ethylene glycol tetraacetic acid&#160;(EGTA) and 0.5% Triton X-100).</li>
	</ol>
	</li>
</ol>
3. Fixation
<ol>
	<li>Aspirate PBSTx and incubate for 15 min with 3% paraformaldehyde (PFA) (Table of Materials) at room temperature, followed by three to four washes with 1x PBS. The fixed cells can be kept at 4 &#176;C until further steps.&#160;&#160;&#160;&#160; 
	&#8203;CAUTION: PFA is highly toxic and carcinogenic. Avoid contact with skin, eyes, and mucous membranes. Perform all the steps with PFA in a fume hood, and dispose of the materials properly.</li>
</ol>
4. Permeabilization and blocking
<ol>
	<li>After fixation, permeabilize the cells using 0.5% PBSTx on ice for 5 min. Use enough volume to cover the entire coverslip (typically between 500 &#181;L and 1 mL).</li>
	<li>Wash the cells three to four times with 0.2% PBST (1X PBS + 0.2% Tween-20) at room temperature. One mL of PBST is enough for washing each well. Perform the washes back-to-back without any incubations.</li>
	<li>Aspirate the PBST and block the samples using 5% bovine serum albumin, BSA (Table of Materials) made in 1x PBS (blocking buffer) for 30 min at room temperature.</li>
</ol>
5. Immunostaining with the IdU antibody
<ol>
	<li>For immunostaining, prepare a humidified chamber (wet paper towel on a flat-bottom Tupperware). Cover the lid of the 24-well plate with parafilm, place in the humidified chamber, and lay down the coverslips on the plate lid.</li>
	<li>Prepare the anti-BrdU primary mouse antibody (Table of Materials) by diluting it 1:200 in the blocking buffer from step 4.2. The anti-BrdU antibody has previously been shown to detect IdU.</li>
	<li>Add 60 &#181;L of IdU antibody dilution on the top of the coverslips. Incubate the coverslips for 1 h at 37 &#176;C.
	<ol>
		<li>Alternatively, use less antibody solution if the coverslips are flipped onto a drop (20-25 &#181;L) of 1:200 antibody dilution pipetted onto parafilm. This also decreases the likelihood of the solution drying out during the incubation.</li>
	</ol>
	</li>
	<li>After the 1 h incubation, aspirate the primary antibody. Return the coverslips back to a 24-well plate, and wash them four times with 0.2% PBST.</li>
	<li>For the secondary antibody, use the same humidified chamber described in step 5.1. Dilute anti-mouse conjugated secondary antibody (Table of Materials) in the blocking buffer (1:200). Add 60 &#181;L of secondary antibody to the coverslips, and incubate at room temperature in the dark for 1 h.This secondary antibody is light-sensitive.</li>
	<li>Aspirate the secondary antibody. Return the coverslips back to the 24-well plate, and wash four times with 0.2% PBST.</li>
	<li>Label microscope slides, and mount the coverslips on the slides with 4&#39;,6-diamidino-2-phenylindole&#160;(DAPI) mounting medium (Table of Materials). Store slides in the dark at room temperature for 24 h. The 24 h incubation is recommended if the mounting medium needs to be cured or hardened.&#160; 
	NOTE: The slides can then be stored in 4 &#176;C before being imaged on a fluorescence microscope. A representative image is shown in&#160;Figure 3.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% Paraformaldehyde (PFA)</td>
			<td>Fisher Scientific</td>
			<td>NC0179595</td>
			<td>10 g sucrose + 100 mL 10X PBS + water to make volume to 925 mL. Add 75 mL 40% Methanol free PFA, mix, and make aliquots of 50 mL before storage 
			Storage: Store in -20 &#176;C</td>
		</tr>
		<tr>
			<td>5-iodo-2&#39;-deoxyuridine (IdU)</td>
			<td>Sigma Aldrich</td>
			<td>I7125-5G</td>
			<td>MW = 354.10 g/mol.For 10 mM stock: dissolve 3.541 mg IdU to 1 mL 1 N liquid ammonia</td>
		</tr>
		<tr>
			<td>Anti-BrdU antibody</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-mouse Alexa Fluor Plus 488 secondary antibody</td>
			<td>Thermo Scientific</td>
			<td>A32766</td>
			<td>Light sensitive - keep in dark</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td>Made by adding specific mass to volume of PBS 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Circular Cover Glass&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72230-01</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td>Growth Media: RPMI supplemented with L-glutamine, 0.01 mg/mL bovine insulin; fetal bovine serum to a final concentration of 20% and 1X Pen Strep 
			Storage: Freezing Media: growth media + 5% DMSO and stored in -80 &#176;C</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>Sigma Aldrich</td>
			<td>P4832-50ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>Thermo Scientific</td>
			<td>P36962</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Water, sterile-filtered</td>
			<td>Sigma Aldrich</td>
			<td>W3500-6X500ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

Source: Ramakrishnan, N. et al., <a href="https://app.jove.com/v/64920">Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence.</a>&#160;J. Vis. Exp. (2023)
The video demonstrates an immunofluorescence-based approach for quantifying replication stress in cancer cells. Hydroxyurea-treated cells produce single-stranded DNA, detected through immunofluorescence. The count of green fluorescence foci indicates the level of replication stress in cancer cells.

<img alt="Figure 1" src="/files/ftp_upload/22136/22136_Figure1.jpg" /> 
Figure 1: IdU labeling schematic.&#160;Cells are pulsed with IdU for two population doublings. If any drug treatment is needed, it should be administered after the first population doubling in the presence of IdU.
<img alt="Figure 2" src="/files/ftp_upload/22136/22136_Figure2.jpg" /> 
Figure 2: Comparison of the foci signal...

NOTE: The ovarian cancer cell line, OVCAR3, was used in these steps, but this protocol is broadly applicable to multiple other cell lines, including those ...

Begin with cancer cells carrying IdU, a synthetic substitute of thymidine nucleotide, already integrated into their DNA. When these cells encounter replication stress, their DNA synthesis halts, revealing IdU within exposed single-stranded DNA regions.
Fix the cells and add detergent molecules to make the cell membrane permeable.
Add BSA to block non-target antibody binding sites.
Introduce anti-IdU antibodies that interact with IdU-labeled single-stranded DNA.
Add green fluorophore-labeled secondary antibodies that target anti-IdU antibodies. Remove the unbound antibodies.
Place the coverslip on a slide containing a mounting medium with a fluorescent dye &#8212; that stains the nuclei.
Under a fluorescence microscope, observe the blue nuclei.
The green fluorescence foci represent antibodies binding to the single-stranded DNA.
Count the number of foci to quantify the level of replication stress.

40 Views. Un metodo basato sull'immunofluorescenza per quantificare lo stress di replicazione nelle cellule tumorali

Video: Un metodo basato sull'immunofluorescenza per quantificare lo stress di replicazione nelle cellule tumorali - Experiment

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

Microscopia di immunofluorescenza di γH2AX e 53BP1 per analizzare la formazione e la riparazione di rotture a doppio filamento del DNA

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

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Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Visualizzazione delle proteine di riparazione del danno al DNA negli organoidi del cancro ovarico derivati dal paziente tramite saggi di immunofluorescenza

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and concentration of the nanomaterials, the time of exposure, and the exposed cell type. Several established methods have been used to elucidate how endogenous/exogenous agents impact global replication. However, replicon-level assays, such as the DNA fiber assay, are imperative to understand how these agents influence replication initiation, terminations, and replication fork progression. Knowing this allows one to understand better how nanomaterials increase the chances of mutation fixation and genomic instability. We used RAW 264.7 macrophages as model cells to study the replication dynamics under graphene oxide nanoparticle exposure. Here, we demonstrate the basic protocol for the DNA fiber assay, which includes pulse labeling with nucleotide analogs, cell lysis, spreading the pulse-labeled DNA fibers onto slides, fluorescent immunostaining of the nucleotide analogs within the DNA fibers, imaging of the replication intermediates within the DNA fibers using confocal microscopy, and replication intermediate analysis utilizing a computer-assisted scoring and analysis (CASA) software.

The methodology assists in the analysis of variation in DNA replication dynamics in the presence of nanoparticles. Different methodologies can be adopted based on the cytotoxicity level of the material of interest. In addition, a description of the image analysis is provided to help in DNA fiber analysis.

An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells

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An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells